View
8
Download
0
Category
Preview:
Citation preview
The CDK9 inhibitor, alvocidib, potentiates the non-clinical activity of azacytidine or decitabine in an MCL-1-dependent fashion, supporting clinical exploration of a decitabine and alvocidib combination
I. Abstract III. Results
Wontak Kim, Clifford J. Whatcott, Adam Siddiqui-Jain, Stephen Anthony, David J. Bearss, and Steven L. WarnerTolero Pharmaceuticals, Inc., Lehi, UT
II. Background
IV. Conclusions
Tolero Pharmaceuticals, Inc., 3900 N Traverse Mtn Blvd, Suite 100 Lehi, UT, 84043 Presented at the 60th ASH Meeting, Dec. 3, 2018, San Diego, CA Scan here to download a copy of our poster:
The hypomethylating agents (HMAs) azacytidine and decitabine exert biological activity viatwo distinct mechanisms, namely, DNA damage and inhibition of DNA methyltransferases.Azacytidine and decitabine are indicated in the treatment of patients with myelodysplasticsyndromes (MDS). As a result of DNA methyltransferase inhibition, it is hypothesized thatHMAs may function by inducing re-expression of key pro-apoptotic proteins such as NOXA,which sequesters the anti-apoptotic protein MCL-1, preventing its association with themitochondrial pore-forming proteins BAX/BAK. Activity of the potent CDK9 inhibitor,alvocidib, is largely driven by targeting of CDK9-dependent MCL-1 expression. Alvocidib isunder active clinical investigation, but has also has demonstrated high complete responserates in newly diagnosed AML patients, particularly when administered as part of acytarabine and mitoxantrone containing regimen (ACM regimen). Given the dualNOXA/MCL-1-targeting ability of combining alvocidib and azacytidine or decitabine, thecombination may synergize therapeutically in the treatment of non-clinical models of AMLor MDS by means of transcriptional induction of NOXA and repression of MCL-1 expression.
Cell viability and induction of apoptosis was assessed following treatment with alvocidib,azacytidine, and decitabine in cells using the Celltiter-Glo and Caspase-Glo assays. Geneexpression changes following treatment were assessed using quantitative RT-PCR. Proteinexpression changes with treatment were also measured using standard immunoblottingtechnique. To assess the in vivo anti-tumor activity of these compounds, xenograft studiesin the MOLM13 and additional models of MDS, exploring sequencing and scheduling ofalvocidib administration with HMAs, were performed. Treatment of AML cell lines withalvocidib inhibited both mRNA and protein expression of MCL-1 in a time andconcentration-dependent fashion. Pre-treatment of cells with alvocidib, to repress MCL-1expression prior to azacytidine treatment, reduced the azacytidine cell viability EC50 morethan 2.5-fold, from 1.8 µM to 0.6 µM in MV4-11 cells. The alvocidib/azacytidinecombination also resulted in synergistic increases in caspase activity relative to either singleagent within the combination, at multiple dose levels. The combination of azacytidine ordecitabine with alvocidib was active in the MOLM13 xenograft model, yielding up to 65.7 or91.1% tumor growth inhibition (%TGI) in the azacytidine or decitabine combination,respectively. Taken together, the in vitro and in vivo studies indicated that decitabine wasmore effective at re-expressing NOXA and potentiating alvocidib activity compared toazacytidine.
These non-clinical data suggest that an alvocidib/HMA combination may constitute a viabletherapeutic regimen whose rationale focuses on hypertargeting of NOXA/MCL-1. Based onthese non-clinical results, a Phase 1b/2 clinical study of alvocidib administered in sequenceafter decitabine in patients with intermediate to high risk MDS is being conducted (Zella102). Patients will be enrolled in cohorts of 3-6 patients with decitabine administered as a 1-hour IV infusion daily on days 1 to 5 at a dose of 20 mg/m2 followed by a single alvocidibtreatment on day 8 as a loading dose over 30 minutes followed by a 4-hour infusion.Treatment will be repeated every 28 days until disease progression or unacceptable toxicity.Enrollment will include MDS patients (Phase 1b) with previously untreated MDS andpatients who received fewer than six (6) cycles of previous HMAs, as well as (Phase 2)untreated patients with de novo or secondary MDS. The primary objective is to determinethe maximum tolerated dose and recommended Phase 2 dose of alvocidib whenadministered in sequence with decitabine. Key Phase 2 endpoints will include completeresponse rate and improvement in transfusion dependency.
Figure 1: Proposed synergy between alvocidib and HMAs
• Alvocidib suppresses MCL-1 expression in a dose-dependent fashion invitro
• Alvocidib clinical activity correlates with NOXA BH3 profiling
• HMAs induce NOXA re-expression in cultured cells
• HMAs induce NOXA re-expression in xenograft models
• Combination of HMAs and alvocidib increased apoptosis in cultured cells
• Sequential dosing, and subsequent NOXA re-expression, are required forincreased apoptosis
• HMAs synergize in xenograft models for MDS/sAML
Figure 10: Clinical trial design (TPI-ALV-102, NCT03593915)
Abstract 4355
• Hypomethylating agents(azacytidine and decitabine)induce re-expression of keyproteins, including NOXA
• The CDK9 inhibitor, alvocidib,suppresses expression of MCL-1
• Increased NOXA expression mayinduce sensitivity to MCL-1 loss
• Alvocidib and HMA combinationsmay be synergistic in cancermodels, including MDS
Figure 2: Alvocidib suppresses MCL-1 mRNA and protein expression
Alvocidib inhibits the transcription of key anti-apoptotic genes, including MCL-1. Following alvocidibtreatment at various doses, both mRNA (A) and protein (B) levels are decreased in a dose-dependentfashion in the MV4-11 (AML) cell line.
MCL-1
β-actin
Alvocidib - + + + + + ++
BA
HMAs, including decitabine, induce NOXA re-expression. As in vitro models of MDS do not exist, theMOLM13 cell line model for secondary AML (prior MDS) was used. MOLM13 cells were treated withdecitabine at the concentrations and times indicated. Expression of mRNA (A) and protein (B) wereassessed using RT-PCR and standard immunoblotting technique.
Figure 4: HMAs induce NOXA re-expression
NOXA
β-actin
Vehicle 4hr 24hr 48hr 4hr 24hr 48hr 4hr 24hr 48hr
100nM 300nM 1μM
BA
Figure 5: HMAs induce rapid NOXA re-expression in vivo
To assess the re-expression of NOXA in vivo, MV4-11 (AML) tumor bearing mice were treated with asingle dose of decitabine (1 mpk). Tumors were then harvested 24 or 48 hours post dosing. NOXA proteinexpression was assessed using standard immunoblotting technique (A). Normalization (relative to β-actin) of immunoblots was averaged and is shown at right (B). Significant animal-to-animal variation wasobserved in basal NOXA expression. On average, NOXA protein levels increased more than 3-fold with asingle dose of decitabine.
NOXA
β-actin
24 hrs 48 hrs
Decitabine - - + + + +
BA
Figure 6: Azacytidine and decitabine induce NOXA re-expression
NOXA
β-actin
BA
To compare the re-expression of NOXA induced by azacytidine and decitabine, MOLM13 tumor bearingmice were treated with a single dose of azacytidine (1 mpk) or decitabine (1 mpk). Tumors were thenharvested 48 hours post dosing. NOXA protein expression was assessed on pooled samples usingstandard immunoblotting technique (A). Normalization (relative to β-actin) of immunoblots is shown atright (B).
Figure 8: Sequential dosing of alvocidib and HMAs in the MOLM13 model for MDS/sAML
A B
C
Alvocidib, azacytidine, anddecitabine activity were assessed inthe MOLM13 xenograft model.Tumor volumes (A), body weights(B), and animal survival (C) areshown. In each weekly schedule,mice were dosed with alvocidib,qdx2, and/or an HMA, qdx3. Dosesare indicated.
Figure 7: Sequential dosing of alvocidib and HMAs synergize to induce apoptosis
Cell viability and caspase activity in cells treated with alvocidib and azacytidine. (A) Cell viability wasassessed (Celltiter-glo) in MV4-11 cells treated with alvocidib (80nM) and various concentrations ofazacytidine. Synergistic anti-proliferative activity was observed in the combination treatment. Caspaseactivity (Caspase-glo) was assessed in MOLM13 cells treated concurrently (B) or in sequence (C). Cellswere treated for 24 hrs with each compound, or a total of 48 hrs with the combination. Dose-dependentsynergy was observed in MOLM13 cells with increasing concentrations of decitabine.
A B C
D
Dose level Days 1-5, Decitabine Day 8, Alvocidib
1-hr IV infusion 30-min bolus 4-hr IV infusion
-1 20 mg/m2 10 mg/m2 10 mg/m2
1 20 mg/m2 20 mg/m2 20 mg/m2
2 20 mg/m2 30 mg/m2 30 mg/m2
3 20 mg/m2 30 mg/m2 45 mg/m2
4 20 mg/m2 30 mg/m2 60 mg/m2
Phase Ib (up to 24 pts)To determine the MTD and RP2D of alvocidib administered in sequence after decitabine in patients with previously untreated MDS and patients with MDS who have received <6 cycles oftreatment with HMAs
Phase II (up to 25 pts)To determine preliminary
efficacy and anti-MDS activity of
alvocidib administered in sequence after
decitabine in untreatedpatients with de novo
or secondary MDS
Efficacy endpointsAssess preliminary
efficacy, as determined by response rate,
duration of response, hematological
improvement, rate oftransfusion independence,
time to acute myeloid leukemia (AML), and overall survival (OS).
Figure 9: Aggressive daily dosing of alvocidib and decitabine in the MOLM13 model
To explore the potential of more aggressive dosing of alvocidib and decitabine in a model for MDS/sAML,MOLM13 tumor bearing mice were treated. Doses and schedule are shown above. Tumor volumes (A) andbodyweights (B) are shown following treatment. As a single agent, alvocidib achieved tumor growthinhibition (%TGI) of 75.8. Decitabine achieved a %TGI of 58.6 as a single agent. The combination achieveda %TGI of 95.8.
BA
Figure 3: Alvocidib shows clinical activity in secondary AML
The ACM (alvocidib + cytarabine + mitoxantrone) regimen has shown activity in clinical studies ofsecondary AML patients. (A) CR/CRi rate for treatment groups was assessed in secondary AML, showing asignificant improvement in CR rates in ACM treated patients. (B) Mitochondrial profiling was performedon pre-treatment bone marrow samples (n = 24) and correlated with response using Mann-Whitney testsfor each mimetic peptide. Patient responses to ACM treatment correlate with NOXA priming in AMLpatients.
A B
Recommended